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Short-term toxicity to fish

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Reference
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11-05-2018 to 03-09-2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.
Qualifier:
according to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
inspected: July 2016; signature: January 2017
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Definitive test: nominal concentrations: 0 (control), 1.0, 1.8, 3.2, 5.6 and 10.0 mg/L
Spacing factor (1.78) determined from range-finding test and physico-chemical properties of the substance.
Controls: test water without test item but treated in the same way as the test item solutions.
- Sampling method: Single samples for analysis were taken from the control and all test concentrations at the start of the test and every 24 hours thereafter in old and new solutions until the end of the test (t=96h)
- Sample storage conditions before analysis: Samples were used on day of sampling and analysed as soon as possible after their preparation.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Stock preparation: The mixing vessel was a cylindrical glass bottle sealed with screw cap and fitted with a drain port near the bottom for drawing off the saturated stock solution. The volume of the mixing vessel was approximately 10 L. A magnetic stirring bar was placed in the vessel and approx. 10 L test water was added. Then an excess of the test item (approx. 1000 mg) was carefully added directly to the surface of the test water. The mixing vessel was thereafter closed immediately. The mixing was initiated with the vortex in the centre extending maximally around 10% vessel depth from the top to the bottom of the vessel. After ca. 24 hours of gentle stirring in the dark at room temperature (at t=0h; the mixing lasted 48 hours), the contents of the vessel were allowed to stand undisturbed for at least 1 hour before use. The first 100 mL were discarded via the drain port. This was subject to analysis. The stock solution was diluted with test water as necessary to obtain the required test concentrations into 5 L volumetric flasks based on measured concentration of the stock solution. Each flask was inverted several times before filling the test vessels (with minimal headspace) before introduction of the test organisms.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable.
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): stock-solution: ca. 14 mg/L t=0h and ca. 6 mg/L (in range finder); final test solutions: nominal concentrations: 0 (control), 1.0, 1.8, 3.2, 5.6 and 10.0 mg/L
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): None. Turbidity testing indicated absence of undissolved test item.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Zebra fish
- Strain: Not reported
- Source: Recognised supplier (listed in full study report)
- Age at study initiation (mean and range, SD):
- Length at study initiation (length definition, mean, range and SD): mean: 29.8 mm (SD; range not stated)
- Weight at study initiation (mean and range, SD): mean: 0.253 g (SD; range not stated)
- Method of breeding: Not reported
- Maintenance of the brood fish: Not reported

ACCLIMATION
- Acclimation period: > 7 days.
- Acclimation conditions (same as test or not): Yes
- Type and amount of food during acclimation: Flaked food or live food (such as brine shrimp nauplii).
- Feeding frequency during acclimation: Daily (feeding ceased 24 hours before test initiation).
- Health during acclimation (any mortality observed): None.

QUARANTINE (wild caught)
- Duration: Internal quarantine at least 12 days after delivery.
- Health/mortality: None.

FEEDING DURING TEST
- Food type: No.
- Amount: Not applicable.
- Frequency: Not applicable.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
none.
Hardness:
Between 10 and 240 mg/L (expressed as CaCO3)
Test temperature:
23 ± 2 ºC (actual 22.7 - to 23.2 ºC)
pH:
7.57-8.03 (fresh media); 6.84-8.10 (expired media)
Dissolved oxygen:
8.22-8.90 mg/L (fresh media); 5.08-6.40 mg/L (or 59.1 to 74.6% ASV) (expired media)
Salinity:
not applicable.
Nominal and measured concentrations:
Range Finding Test:
Nominal Concentrations: 0 (control), 0.32, 1.00, 3.20, 10.00, 14.00 mg/L (maximum solubility)
Geometric Mean Measured concentrations: 0 (control), 0.88, 2.87, 10.02, 14.18 mg/L
Definitive Test:
Nominal test concentrations: 0 (control), 1.00, 1.80, 3.20, 5.60, 10.00 mg/L
Geometric Mean Measured concentrations: 0.89, 1.60, 2.98, 5.14, 9.41, mg/L
Percent of nominal: 90%, 90%, 95%, 93% and 95%, respectively.
Details on test conditions:
TEST SYSTEM
- Test vessel: 3.5 L glass.
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: ca.3.5 L fill volume test media per test vessel (minimum headspace ca. 1 cm)
- Aeration: no
- Renewal rate of test solution (frequency): 24 h
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): Not applicable
- Biomass loading rate: < 1.0 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Ultrapure water.
- Total organic carbon: Not reported
- Particulate matter: Not reported
- Metals: Not reported
- Pesticides: Not reported
- Chlorine: Not reported
- Alkalinity: Not reported
- Ca/mg ratio: Not reported
- Conductivity: < 10 μS/cm
- Culture medium different from test medium: No. Reconstituted water in accordance with OECD TG 203.
- Intervals of water quality measurement: pH, nitrate and nitrite concentration, ammonia concentration, total water hardness and dissolved oxygen: once a week. Temperature: every day.

OTHER TEST CONDITIONS
- Adjustment of pH: No.
- Photoperiod: 16 h light / 8 h darkness
- Light intensity: white fluorescent lamp, intensity not reported

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : mortality and visible abnormality recorded at least the following: 24 h, 48 h, 72 h and 96 h.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.78 (Definitive)
- Range finding study: yes (static)
- Test concentrations: Range Finder: 0 (control), 0.32, 1.00, 3.2, 10.0 and 14.00 mg/L (maximum solubility) ; Definitive: 0 (control), 1.0, 1.8, 3.2, 5.6, 10.0 mg/L
- Results used to determine the conditions for the definitive study: Yes. Static test indicated not possible to maintain test item concentrations during exposure. Results: At 1 mg/L and 10 mg/L: 0% cumulative mortality at 96 h time point. At 50 mg/L and 100 mg/L: 100% cumulative mortality at 24 through 96 h time points.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
3.601 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other: (95% CL: 3.086-4.202 mg/L)
Details on results:
- Behavioural abnormalities: See table 2
- Observations on body length and weight: Not reported
- Other biological observations: Not reported
- Mortality of control: 0%
- Other adverse effects control: No abnormalities observed in the control.
- Abnormal responses: Sub leathal responses are reported in table 2
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None. Turbidity measurements indicated the absence of any undissolved test item.
- Effect concentrations exceeding solubility of substance in test medium: Not applicable
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Mortality: Not reported
- LC50: 195.554 mg/L (95% CL - mg/L) in 24 h
- Other: In agreement with laboratory historical database
Reported statistics and error estimates:
Evaluation of the effects was based on measured concentrations using ToxRat Professional Software to determine effective concentrations using Probit analysis. LCx was determined by Spearman-Karber procedures (for survival at 24, 48 and 72h) and Trimmed Spearman-Karber (for survival at 24h and 96h). Results provided in the full study report. The 95% confidence intervals were determined, as appropriate.

It was not possible to derive confidence limits using a Probit-regression due to limitations in the data set at t=24h and t=96h, it was therefore decided to use a more statistically robust non-parametric test with less required assumption of the data set (the trimmed Spearman-Karber method) for estimation of the 24 and 96-LC50 values including the 95% confidence interval.

For 48 and 72 hours, two concentrations in the series which directly succeed one another caused 0 and 100 % mortality. Therefore, no regression could be applied. The LC50 was therefore calculated as A+B/2, and the confidence limits became concentration A and B, where A is the immediate concentration that causes 0 % mortality succeeding the concentration which results in 100 % mortality, and B is the first concentration causing 100 % mortality. Reference: Rand, G. M. and Petrocelli, S. R. (1985)
Sublethal observations / clinical signs:

Table 1.0 – nominal and measured concentrations

Nominal

concentration

mg/L

Expected

nominal

concentration

in test item

(mg/L

t=48h fresh

t=72h old

Relative loss to initial value

(t=48hfresh - t=72hold)

(%)

t=72h fresh

End

t=96h

Relative loss to initial value

(t=72h fresh - t=96)

(%)

Geometric mean measured concentrations

Meas. Conc mg/L

%

nominal

Meas. Conc mg/L

Meas. Conc mg/L

Meas. Conc mg/L

%

nominal

Meas. Conc mg/L

%

nominal

mg/L

%

nominal

Control

0

Abs.

N.A.

Abs.

Abs.

N.A.

Abs.

N.A.

Abs.

Abs.

N.A.

N.A.

N.A.

1.0

0.99

0.97

98

0.84

85

13

1.00

101

0.81

82

19

0.89

90

1.8

1.77

1.73

98

1.44

81

17

1.83

103

1.39

79

24

1.60

90

3.2

3.15

3.08

98

2.73

87

11

3.36

107

3.07

97

9

2.98

95

5.6

5.52

-

-

-

-

-

-

-

-

-

-

5.14

93

10.0

9.86

-

-

-

-

-

-

-

-

-

-

9.41

95

N.A.: not applicable

-: no further analysis was performed for concentrations where there was complete mortality

% = Percent of expected nominal concentration in test item.

Abs.= Absence: concentrations below the LOQ (0.11 mg/L) and the LOD (0.03 mg/L).

 

Table 2.0 - Cumulative mortality

 

Nominal concentration

and corresponding measured concentration (geometric mean)

(mg/L)

Nominal

 

Cumulative Mortality

Control

(0)

M     S

1.0

(0.89)

M     S

1.8

(1.60)

M     S

3.2

(2.98)

M     S

5.6

(5.14)

M     S

10.0a

(9.41)

M     S

t=24h

0      0

0      0

0      0

0      2b

1      1

7     0

24h Total Mortality (%)

0

0

0

0

14.3

100

t=48h

0      0

0      0

0      0

0      3b

7      0

7     0

48h Total Mortality (%)

0

0

0

0

100

100

t=72h

0      0

0      0

0      0

0      7b

7      0

7     0

72h Total Mortality (%)

0

0

0

0

100

100

t=96h

0      0

0      0

0      0

1      6b

7     0

7     0

96h Total Mortality (%)

0

0

0

14.3

100

100

M: Number of dead fish; S: Number of surviving fish but with sublethal effects

a: At t=3h, sublethal effects (fish distributed at the bottom of the aquarium) were observed at 10.0 mg/L ; 3 fish were dead at t=6h and removed from the test vessel.

b: Fish with pronounced sublethal effects (fish distributed at the bottom of the aquarium, with weakening and breathing difficulties).

The initial number of fish in each test vessel was 7.

Validity criteria fulfilled:
yes
Conclusions:
The 96 hour LC50 for the test item to Danio rerio was determined to be 3.601 (C.I. 3.086 - 4.202) mg/L based on geometric mean measured concentrations.
Executive summary:

The acute toxicity of the test item to Zebra fish (Danio rerio) was determined in a 96 hour closed semi-static test according to OECD TG 203 and EU Method C.1 under GLP. Based on the results of preliminary range finding testing, the nominal concentrations of 0 (control), 1.0, 1.8, 3.2, 5.6 and 10.0 mg/L with a geometric series factor of 1.78 and minimal headspace which and were selected for exposure to groups of seven fish. One control containing no test item was also performed.The test total duration was 96 hours and test solutions were renewed on a daily basis. The mortality of the fish was determined by visual observation after 24, 48, 72 and 96 hours. Samples taken from the control and all test concentrations were analysed at the start of the test and every 24 hours thereafter in old and new solutions. The test item was renewed at 24 hour intervals within a 96-hour semi-static test system. The actual concentrations were verified by measurement of the test item in the nominal loading rates of 0 (control), 1.0, 1.8, 3.2, 5.6 and 10.0 mg/L at 0h, 96h and 24h,48h, 72h of new and old solutions (after and before the renewal).Analytical results showed that test item levels were overall maintained within 80% of the nominal (and initial) concentrations throughout the duration of the test, except for the first day of the test where measured concentrations were slightly lower than 80%. Therefore, the evaluation of the effects was based on the geometric means of the measured concentrations: 0.89, 1.60, 2.98, 5.14 and 9.41 mg/L.After the 96-hours exposure, mortality was 0% at 0.89 and 1.60 mg/L, 14.3% at 2.98 mg/L, and 100% at 5.14 mg/L and 9.41 mg/L. Measurements were performed by High Performance Liquid Chromatography (HPLC-DAD). All relevant validity criteria were fulfilled for the test. The relevant LC50 were: 24h: 6.406 (C.I. 5.502 – 7.459) mg/L, 48h: 4.060 (C.I. 2.980 – 5.140) mg/L, 72h: 4.060 (C.I. 2.980 – 5.140) mg/L based on geometric mean measured concentrations. The 96 hour LC50 was 3.601 (C.I. 3.086 – 4.202) mg/L based on geometric mean measured concentrations.

Description of key information

LC50-96h (fish) = 3.601 mg/L (C.I. 3.086 – 4.202 mg/L) based geometric mean measured concentrations, 96-hour, freshwater, OECD TG 203, 2018

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
3.601 mg/L

Additional information

Key data : OECD TG 203, 2018 : The acute toxicity of the test item to Zebra fish (Danio rerio) was determined in a 96 hour closed semi-static test according to OECD TG 203 and EU Method C.1 under GLP. Based on the results of preliminary range finding testing, the nominal concentrations of 0 (control), 1.0, 1.8, 3.2, 5.6 and 10.0 mg/L with a geometric series factor of 1.78 and minimal headspace which and were selected for exposure to groups of seven fish. One control containing no test item was also performed.The test total duration was 96 hours and test solutions were renewed on a daily basis. The mortality of the fish was determined by visual observation after 24, 48, 72 and 96 hours. Samples taken from the control and all test concentrations were analysed at the start of the test and every 24 hours thereafter in old and new solutions. The test item was renewed at 24 hour intervals within a 96-hour semi-static test system. The actual concentrations were verified by measurement of the test item in the nominal loading rates of 0 (control), 1.0, 1.8, 3.2, 5.6 and 10.0 mg/L at 0h, 96h and 24h,48h, 72h of new and old solutions (after and before the renewal).Analytical results showed that test item levels were overall maintained within 80% of the nominal (and initial) concentrations throughout the duration of the test, except for the first day of the test where measured concentrations were slightly lower than 80%. Therefore, the evaluation of the effects was based on the geometric means of the measured concentrations: 0.89, 1.60, 2.98, 5.14 and 9.41 mg/L.After the 96-hours exposure, mortality was 0% at 0.89 and 1.60 mg/L, 14.3% at 2.98 mg/L, and 100% at 5.14 mg/L and 9.41 mg/L. Measurements were performed by High Performance Liquid Chromatography (HPLC-DAD). All relevant validity criteria were fulfilled for the test. The relevant LC50 were: 24h: 6.406 (C.I. 5.502 – 7.459) mg/L, 48h: 4.060 (C.I. 2.980 – 5.140) mg/L, 72h: 4.060 (C.I. 2.980 – 5.140) mg/L based on geometric mean measured concentrations. The 96 hour LC50 was 3.601 (C.I. 3.086 – 4.202) mg/L based on geometric mean measured concentrations.

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